The long term objective of this work is to understand the mechanism of nuclear pre-mRNA splicing, a fundamental process that takes place in all eukaryotic cells and is a required step for the expression of most cellular and viral protein-coding genes. To this end, biochemical and molecular biological approaches will be employed for a detailed analysis of the structure and function of selected human protein factors required for splicing. Biochemical methods will also be used to identify, isolate, and characterize novel protein factors required for splicing. Biochemical complementation of selectively inactivated splicing extracts will be used as an assay for purifying these factors in their active states. Recombinant human splicing factor SF2/ASF and human immunodeficiency virus (HIV) or growth hormone pre-mRNAs will be used to study sequence-specific protein-RNA interactions between splicing factors and exonic splicing enhancer elements. The goal is to understand how these interactions can modulate the specificity and efficiency of pre-mRNA splicing. SF2/ASF mutants and related human proteins will also be analyzed to understand the molecular basis of these interactions. Selection and amplification cycling methods will be used to identify the RNA-binding specificity of each domain in these proteins. Physical studies of the structures of these proteins and domains in the presence and absence of target RNA will be initiated. Proteins related to human SF2/ASF will be sought in yeast to allow genetic dissection of the function of this class of proteins and comparative analysis between yeast and metazoan splicing factors. Errors in splicing specificity caused by mutations in intron-containing genes are often the cause of many human genetic diseases. Because the same mutations also affect splicing specificity in vitro, the molecular basis of the mechanisms of splicing specificity is amenable to biochemical analysis. Recent in vitro experiments made use of antisense modified oligonucleotides complementary to cryptic splice sites to correct aberrant splicing associated with beta-thalassemia mutations. In addition, genetic defects in the expression or structure of cellular splicing factors might be associated with inherited diseases or cancer. Inefficient use of splice sites, another aspect of splicing specificity, is an important feature of the life cycle of retroviruses, including HIV. The spliceosome, a multienzyme complex, and its individual components, remain to be explored as potential targets for novel therapeutic agents. In the long term it may be possible to identify or design drugs that change the concentration or biochemical properties of specific spliceosome constituents in such a way as to compensate for decreased or aberrant splicing in mutant genes. Thus, these studies, which are aimed at understanding basic cellular mechanisms of gene expression, may provide the basis for the development of new diagnostic tools and therapeutic agents.